This award will support my transition into the area of stem cell biology. I plan a combination of directed laboratory experience and participation in laboratory and group meetings as well as relevant journal clubs and activities of the Harvard Stem Cell Institute. In short, I will take full advantage of the robust and diverse stem cell community at Harvard. Under Dr. Scadden's mentorship, I will extend our previous work on zebrafish hematopoietic stem cells and begin the study of pathways that regulate the balance between stem cell self renewal and the generation of progenitor cells. The proposal extends our recent discovery of a way to tag hematopoietic stem cells with fluorescent reporter dyes, transplant them into irradiated host zebrafish, and achieve long-term multi-lineage engraftment. We will define pathways that regulate hematopoietic stem cell self renewal by engineering transgenic zebrafish that over-express candidate regulators. Stem cells will then be isolated from wild type and transgenic fish and stem cell repopulating ability will be assessed in irradiated host fish. We will: 1) develop a model in zebrafish to carry out stem cell transplants, limiting dilution and competitive repopulation assays using hematopoietic stem cells (HSCs) from the whole kidney marrow of zebrafish carrying the CD41-GFP transgene;2) construct transgenic fish lines using the Gal4/UAS system to study the effect of the Wnt, Notch and hedgehog signaling pathways on hematopoietic stem cell self renewal and proliferation;3) test the effect of over-expressing or inhibiting components the Wnt, Notch and Hedgehog pathways on stem cell proliferation. Pathways will be activated or inhibited by varying periods of heat shock followed by harvest of stem cells, enrichment by flow sorting and transplantation into irradiated recipients. We will analyze the effects of (3-catenin (Wnt pathway), hedgehog, intracellular Notch as well as the Wnt inhibitor Axin, two Notch inhibitors - dominant negative forms of Xenopus suppressor of hairless (dnXsu(H)) and a dominant negative form of mastermind like gene 1 (dnMALMI) and two small molecule hedgehog inhibitors cyclopamine and CUR 61414. We will also test the effects of Bone morphogenetic protein 4 (BMP) and its inhibitor Noggin as BMP activity is in part regulated via the hedgehog pathway. We hope to gain information that can be used in future studies to find small molecule activators of relevant pathways and test their efficacy in experimental models and humans. (End of Abstract)